Method of isolating gastric secretion inhibitory substance, and the enzyme gastricsin, and the resulting products



United States Patent 6 3,026,251 METHOD OF ISOLATING GASTRiC SECRETEON INHEBITORY SUBSTANCE, AND THE ENZYME GASTRICSZN, AND TE RESULTHNG PRODUCTS Stewart G. Wolf, in, Ranwel Caputto, and Raul E. Trucco, )lrlahoma City, kla., assignors to Research Corporation, New York, N.Y., a corporation of New York No Drawing. Filed Feb. 3, 1959, Ser. No. 799,808

15 Claims.

This invention relates to the production of a valuable therapeutic substance from mammalian material and particularly to the preparation of a gastric secretion inhibitor substance from tissue containing it, and especially to the isolation of said inhibitor substance from animal or human sources. This invention relates also to the production from said animal or human sources of a newly discovered proteolytic enzyme of gastric origin, namely, gastricsin.

Medical science has been aware for a number of years that the intravenous administration of normal human gastric content to animals produces a highly significant inhibition of both the quantity and acidity of the gastric juice of the animals so injected. It was further recognized that the stomach content of patients sufiering rom achlorhydria due to pernicious anemia or other causes, has greater inhibitor potency than normal human stomach content and that the gastric secretion inhibitory efiect could also be obtained in animals by administration of various animal extracts of salivary or gastric origin. Further development of the potentialities of this specific inhibitor effect as a research tool and as a medical weapon against for example, peptic ulcer, has not been realized and in fact has been thwarted due to lack of a sufiicieritly potent extract containing the inhibitor substance. This lack is in turn due to the absence of a suitable process for isolating the inhibitor substance from animal, including human, material containing it.

Accordingly, this invention provides a method of isolating the inhibitor substance and for preparing a valuable and potent extract of it capable of convenient use in the field of medical science.

The process of this invention makes it possible for the first time to separate from the large molecular (ie high molecular weight) components of gastric content, such as pepsin, blood group A substance, various mucopolysaccharides and various proteins, etc., a fraction of the desired components which is responsible for a specific effect when administered to humans or animals, namely, the inhibition of gastric acid secretion.

The inhibitor itself is a water-soluble, non-dialyzable protein or mucoprotein which occurs in the gastric juice. Its effect is specific to quantity of secretion and acid, but it does not alter the secretion of pepsin or other nitrogen containing substances. The activity of the desired inhibitor is not destroyed by boiling for 15 minutes but it is destroyed by incubation with whole gastric juice for one-half hour at a pH of 1 and at 37 C.

A further application of the process of this invention, with modifications to be described hereinbelow, results in the isolation of a pure crystalline gastric proteolytic enzyme newly discovered by and identified by the present inventors, which we have called gastricsin. The production of gastricsin is a unique application of, and forms an essential part of, the present invention and because the isolation of gastricsin preferably and most conveniently proceeds concurrently with the isolation of the gastric secretion inhibitory substance, it is described and claimed herein.

Therefore, it is an object of this invention to provide 3,025,251 Patented Mar. 20, lQfiZ a process for the fractionation of material containing gastric secretion inhibitory substance and gastricsin.

It is a further object of this invention to provide a chromatographic fractionation process whereby the isolation of gastric secretion inhibitory substance and of gastricsin may be readily accomplished.

Another object of this invention is to provide a chromatographic resin column extraction procedure for gastric juices which results in the isolation therefrom of the desired gastric secretion inhibitory substance and pure crystalline gastricsin.

A further important object of this invention is to provide a potent extract containing the gastric secretion inhibitory substance suitable for therapeutic use.

A specific object of this invention is to provide a method for producing a fraction of gastric juice which has a gastric secretion inhibitory potency of the order of approximately five times its weight of whole gastric ince.

Still another object of this invention is to provide a method for isolation and purification of the newly discovered enzyme, gastricsin.

Further objects and advantages will appear from the detailed description and discussion of the invention which follows and which is set forth first in relation to the isolation of the gastric secretion inhibitory substance and secondly in relation to the isolation of gastricsin.

The process of this invention comprises generally the steps of obtaining a crude source of inhibitor such as animal gastric tissue and/or juices, or human gastric juice; separating the macromolecular substances comprising this source material; factionating these substances by chromatrographic procedures such as contacting same with an ion-exchange resin; collecting the various inhibitor substance-fractions thus obtained; and finally purlfying the inhibitor substance-containing fractions so as to provide a dry powdered substance which may be standardized and used, e.g. in aqueous solution, in the management of medical patients.

By the process of this invention an active fraction containing the inhibitor factor can be isolated in a relatively pure form from the initial source. The product of this invention may then be easily reconstituted for use in the treatment of e.g. peptic ulcer patients.

More specifically, the process of this invention comprises preparing a liquid extract of the source material which contains the gastric secretion inhibtor substance, dialyzing this solution or suspension against distilled water to remove the salts and other low molecular weight components and lyophilizing the dialysate to a dry powclot.

The dried powder containing the large molecular components, i.e., the colloid fraction of the dialysis procedure, is then dissolved in a buffered aqueous solution and the insoluble portion removed.

As a feature of the present invention, it has been discovered that the gastric secretion inhibitory substance can be and is selectively adsorbed on a suitably prepared fractionation column of ion-exchange resin and further that by maintaining proper conditions of pH in the column by using suitable buffered eluants, the inhibitor substance is selectively released within a predetermined range of pH values.

The inhibitor substance, therefore, may be collected separately in the fraction eluted within that range, pooled, purified by dialysis, and dried by lyophilization. The resultant dried powder is a relatively pure extract of gastric secretion inhibitor substance having the properties previously mentioned which is many times more potent than the original starting material on a Weight basis. It may be subjected to biological standardization and suitably packaged to supply, upon the addition of a vehicle such as isotonic saline solution, a calculated dose or doses of predetermined potency.

The following description of a preferred embodiment of the process of this invention will serve to illustrate in detail (1) the preparation of a suitable source material, e.g., human gastric juice; (2) the preparation of the chromatographic resin column; (3 the fractionation procedure; and (4) the test procedure for determining the biological activity of the product of the invention.

EXAMPLE I Collection and Storage of Gastric Juice Preparation of the Resin Column Fractionation of the whole dried gastric juice is accomplished as described hereinafter, with a suitable ionexchange resin such as Amberlite IRC-50 (XE-64) obtained from the Rohm and Haas Company, Philadelphia, Pennsylvania. This is a carboxylic acid type cation exchange resin which is weakly acidic and in hydrogen form. It is granular and of 100400 mesh as purchased. The resin is treated as follows in order to prepare it for the fractionation step which follows:

A mass of resin having a 200-400 mesh range is prepared. To 1.5 kilos of this resin, 3.5 liters of water are added and the suspension is stirred for 20 minutes. After a settling period of 30 minutes, any foam on the surface is removed and the supernatant suspension is withdrawn. The settling process is repeated four or five times with 2-liter portions of water until the supernatant liquid is clear after about 15 minutes of settling. The resin is air-dried on a Buchner funnel. The resin filter cake is added to 4 liters of acetone and stirred for 3 hours. The resin is filtered and washed with acetone (about 8 liters) until the filtrate is clear. The air-dried resin is suspended in water, stirred until all bubbles are eliminated and the last traces of acetone are removed by washing the filter with 24 liters of water.

The resin is cycled once through the sodium form before use, as follows: To the resin in 5 liters of water, 560 gm. of sodium hydroxide (in the form of a 40 percent solution) are added over a period of 30 minutes. The pH of the suspension will rise to about 11. Stirring is continued until evolution of heat has subsided (3 hours), and the sodium salt of the resin is then washed by decantation with five 2-liter portions of water. The resin is washed on a filter with water (about 12 liters) until the filtrate has a pH of about 10. The resin is converted to the acid form by passing liters of 3 N hydrochloric acid through the filter over a 4 hour period, and is finally washed with 6 liters of Water.

A suspension in water of 100 gm. of the thus-treated resin (now in the acid form) is poured into a chromatographic tube of 4.5 cm, diameter which gives a column cm. high. The column is then equilibrated with 0.2 M citrate buffer at pH 3.0 (formula given below) by passing the bufier through the column until the efiiuent reaches a pH of 3.

Fractionation Procedure Loading of the column-3 gm. of dried gastric juice prepared as described above are dissolved in 120 ml. of

0.2 M citrate buffer of pH 3.0 (see formula below) and the insoluble portion separated by centrifugation at 10,000 g and then removed. The centrifuged solution is poured into the column. 7

Running of the c0lumn.The column is then run from pH 3.0 to pH 8.5 with the following buffers: 0.2 M citrate of pH 3.0; 0.2 M citrate of pH 3.8; 0.2 M citrate of pH 4.2; 0.2 M citrate of pH 4.6; 1.0 M phosphate of pH 5.3; 1.0 M phosphate of pH 6.2; 1.0 M phosphate of pH 7.0; and 1.0 M phosphate of pH 8.5.

The composition and preparation of the buffers are as follows:

0.2 M citrate pH 3.0:

Citric acid gm 42.0 Sodium hydroxide gm 4.4 Sodium chloride gm 13.5 Distilled water liter 1 0.2 M citrate pH 3.8 and pH 4.2:

Citric acid gm 42.0 Sodium hydroxide gm 7.0 Sodium chloride gm 9.6 Distilled water liter 1 0.2 M citrate pH 4.6:

Citric acid gm 42.0 Sodium hydroxide gm 12.0 Sodium chloride gm 2.34 Distilled water liter 1 The substances are dissolved in less than 1 liter of water, then a concentrated solution of sodium hydroxide.

is added to adjust to the desired pH, and finally the volume is adjusted to 1 liter,

1.0 M phosphate pH 5.3:

Sodium monophosphate (i.e.,

The buffers are run through the column in the order specified above. One changes to the buffer of the next higher pH when the pH of the effluent approaches that of the influent buffer. The buffer in the reservoir at the top of the column is removed just to the top of the resin and the next bufier to be used is added carefully to the top of the resin. A few cc. of chloroform (for antiseptic purposes), are added to the containers of the bulfers to keep them saturated While they are running through the column. The rate of flow is about 8 ml./20 minutes. Elution of the protein components is followed by measuring absorption at 280 mu of the eluates in silica cells with a quartz Spectrophotometer.

The fractions are collected in each of the following pH intervals: fraction #1, 1200 ml., from pH 3.0 to 3.9; fraction #2, 1800 ml., from pH 3.9 to 4.6; fraction #3, 2100 ml., pH 4.6 to 5.4; fraction #4, 1700 ml., pH 5.4 to 6.15 and fraction #5, 3200 ml., pH 6.15 to 8.5. Each fraction is pooled separately. These fractions are dialyzed in cellophane tubing against five changes of distilled water at 4 C. in the presence of chloroform. They are then lyophilized and the dried powder stored in a desiccator at 4 C. The weight of each dried fraction is #1, 0.278 gm.; #2 0.595 gm.; #3, 0.715 gm.; #4, 0.156 gm. and #5, 0.480 gm.

The collection of the eluate in more than two separate fractions as described above facilitates the recovery of other components of the gastric material. It may be possible however, where the gastric secretion inhibitor alone is desired, to run the fractionation column from the starting pH more rapidly up to pH 6.15, using an initial buttered eluant of pH 6.15 and begin the collection of the active inhibitor fraction at that point.

Activity Test The inhibitor is assayed for its biological activity in inhibiting gastric secretion of Heidenhain Pouch dogs.

The operated dogs are allowed to stabilize for 3-4 week period before tests are begun; control juice collections are then obtained daily during a four hour period after the dogs have been fed and tests are started only when these collections become fairly constant. The juice drains into rubber bladders attached to the cannula and supported by a sling about the dog. Volume, free and total acidity are measured.

Tests are carried out in the following fashion: Dogs are fed with a test ration and fifteen minutes later injected intravenously with one of the dried fractions which has been dissolved in isotonic saline solution at a concentration of 1 mg./ml., centrifuged and filtered through sterilized glass sintered filter (U.F. Pyrex). The dose of each fraction is uniform, 0.2 mg./kg. of dog weight. For comparative purposes, some dogs are injected with 1 mg./kg. of dog weight of the whole dried gastric juice. Rubber bladders are attached to the cannula fifteen minutes after injection and kept on for four hours. The rubber bladders are then removed, the juice collected and determinations made as to volume, free and total acidity. Rectal temperatures are taken frequently during the postinjection period and the dogs are observed closely for reactions. If a rise in body temperature greater than 1.5" F. occurs or if the dog vornits after the injection the test is discarded.

Results of the Tests weight Analysis of the gastric juices collected following the injections indicates that inhibitory activity is present only in fraction No. 5 (eluted at pH 6.15-8.5) and in the whole dried gastric juice. The normal gastric secretion of the test animals is reduced 50% by the injection of fraction No. 5 and of whole dried gastric juice as shown in Table I. Because the injected dose of whole dried gastric juice is five times as great as that of the fraction No. 5 however, these results show that fraction No. 5 is five times as potent on a weight basis as the whole dried gastric juice.

Furthermore, the lack of inhibitory activity of the other fractions also indicates that substantially all the gastric secretion inhibitory activity is present in fraction No. 5, in a form which is relatively pure and which with relative case may be administered intravenously to obtain the desired efiect of gastric secretion inhibition.

While the above test was conducted on dogs the same relationship between whole dried gastric juice and the active fraction is believed to hold for human administration also. Thus, since the intravenous administration to humans of 0.1 mg./kg. of body weight of whole dried gastric juice has produced evidence strongly suggestive of inhibition, the dose of the gastric secretion inhibitory product of this invention may be stated as approximately 0.02 mg./kg. body weight. The dose, however, must be determined by the circumstances and condition of each patient.

It is noted that under the conditions of the fractionation procedure given above, it has been discovered according to the present invention that the inhibitor substance can be successfully eluted from the column by appropriate buffer solutions within a certain range of pH of the efiiuent, namely, 6.15-8.5. The fraction thus obtained when dialyzed and lyophilized represents only about 10% of the weight of the whole dried gastric juice from which it has been separated. This product containing gastric secretion inhibitory substance is obviously vastly preferable to a crude whole extract of gastric content or of gastric tissue and will find valuable use not only in peptic ulcer patients but in other syndromes involving hypersecretion of stomach acid.

A further advantage and result of the process of this invention is the isolation from human gastric juice of a heretofore unknown and unidentified proteolytic enzyme. This enzyme is here called gastricsin to distinguish it from pepsin, the only previously known proteolytic enzyme of human gastric origin.

This enzyme, gastricsin, is obtained from whole gastric juice in a pure crystalline form by subjecting the gastric juice to the same process steps as described above in Example I, and further purification with ammonium sulphate. Gastricsinis obtained as a homogeneous fraction and is eluted at an efiiuent pH of 4.4. Therefore, it may be isolated during the running of the column in accordance with the above example simply by collecting separately the elute of pH 4.4. Another method for isolating gastricsin is by the further fractionation of the fraction obtained at an eluant pH of 3.9-4.6 (fraction No. 3) in the above Example I by rerunning it through a similar resin column under similar conditions. The gastricsin again will be present in the eluate at pH 4.4 as a homogeneous aqueous fraction.

By either method, the gastricsin is successfully separated from the pepsin which if present is eluted by the process of this invention at the pH range of 3.8 to 4.4. The presence of pepsin in the eluate within this range has been previously demonstrated by the present inventors. (Archives of Biochern, and Biophysics, 66, 155466 (1957).)

The isolation of gastricsin in accordance with this invention is illustrated by the following example.

EXAMPLE II Again referring to the process of Example 1, 2 gm. of a similarly prepared whole dried gastric juice are fractionated in the same manner and an eluant of pH 4.4 measuring 200 ml. is obtained. Following dialysis and lyophilization of this eluant fraction a chromatographically pure product (gastricsin) weighing 36 mg. is recovered.

Crystallization of the Enzyme This product is then crystallized by the following procedure: The dried powder is dissolved in a 12 ml. centrifuge tube containing 2.0 ml. of ice cold distilled Water. The solution is centrifuged at 2,000 r.p.m. for 5 minutes to remove any insoluble material which may be present due to denaturation of the enzyme during lyophilization. The supernatant is poured into another centrifuge tube, put back immediately into an ice bath, and 0.26 gm. of crystalline (NH SO added. The tube is stirred gently until the (NH SO is completely dissolved and then centrifuged at 2,000 r.p.m. for 10 minutes. The clear super natant is decanted and discarded. The precipitate is dissolved with 2 cc. of water and the previous precipitation is repeated. The tube containing the precipitate is returned to an ice bath and 2 ml. of Na-acetate bufier (pH 5.0) is added to dissolve the precipitate. Small portions of crystalline (NH SO are added to the solution and stirred so that the (NH SO goes into complete solution after each addition. This process is stopped at the first indication of protein precipitation. The amount re- 8 changes and modifications may be made in the details of the methods of procedure without departing from the spirit of the invention. For example, although human gastric juice has been specifically used herein, the source quired is approximately 0.2 gm. (NH SO The tube 5 material may be animal gastric juice or tissue such as is then moved to a Water bath (about 20 C.) and left for hog stomach, particularly for commercial exploitations 5 minutes. If the turbidity disappears, very small drops of the process of this invention. Additionally, other inof saturated (N-HQ SQ; solution are added gradually, organic buffers of appropriate pH such as borate may until the turbidity reappears. The solution is stirred be used. Also the rate of flow of the elueates is calculated thoroughly after addition of each drop. The tube is then to ensure complete extraction and may vary within, for moved to 40 C. Water bath and kept in it for minutes. example, and 45 ml./hr. The turbidity usually disappears; when it does not it is cen- The size of the resin column will depend generally trifuged and the insoluble material removed. on the amount of the material to be fractionated and the The tube is placed in a beaker containing 2 liters of resin itself will in accordance with per se known chromatowater at 30 C. Then the beaker is moved into the 4 C. 15 graphic techniques be chosen to supply along with apcold room where after 6-8 hours a white precipitate is propriately chosen buffers, the conditions of pH essential formed. The first precipitate however is mostly nonto fractionation and eluation of the products of the incrystalline material and is removed by centrifugation at a vention as set forth herein. Of. the available apparatus 'low speed (in the 4- C. cold room or in a refrigerated suitable for the fractionation and collection aspect of this centrifuge). The supernatant is left standing at 4 C. invention We refer, for. the sake of brevity, only to the for a period of 20 to 30 hours during which crystals are automatic fraction collecting apparatus disclosed in US. formed. After the crystals have been standing for 23 Patent No. 2,654,522 to Gorham. days the first collection is made by centrifuging at low Therefore, it will be understood that the examples speed. The supernatant which still contained high enzyme given and the details of the procedures set forth herein activity is left standing for more crystals to form. When are intended to be illustrative only are are not intended no significant increase of crystals in the suspension is obto limit the scope of the invention. served, after another 6 hours, drops of saturate ammonium Wh t i laimed i ph 891115011 are added- The Yield of crystalline 1. In a process for isolating the enzyme gastricsin and gastl'lcsm 20% of the chromatosraphically a Water-soluble, protein-containing, gastric secretion in- P hibitory substance from gastric material of animal, in-

y Welght Ylelds, SPeCIfiC actlvities' d activity cluding human, origin, the steps of selectively adsorbing Q Q 0f phromatogfaphicany P and Crystalline the water-soluble non-dialyzable components of said tr1cs1n obtained from preparation described in Example gastric i l o a column of carboxylic acid type II are ShQWn 1n the following Table III cation-exchange resin equilibrated at about pH 3, eluting TABLE II the said column with a series of inorganic buffer solutions of increasing pH values within the range of from about Dry Specific Total 3 to about 8.5, said series containing members having Fractions gi g ggg; approximate pH values of 3, 3.8, 4.2, 4.6, 5.3, 6.2, 7.0, p and 8.5, and each of said members being employed until Freezedfied Gasmemice 2,000 445 100 40 the efiluent pH reaches a value equal to that of said Chromatographic Fractio 36 153.6 member, producing eluate having a pH value gradually Crystals 2 8 increasing from about 3 to about 8.5, collecting separately iDetermined bv method of Anson and Mirsky J Gen the fraction eluted at the pH of about 4.4 and the frac- Physiolq 16 59 (i932) 5 tron eluted at the pH of from 6.15 to about 8.5, recovermg the enzyme gastncsm from said first named fraction, Propemes of the Enzyme and recovering from said second named fraction a con- Properties of gastricsin and its comparison with other centrate comprising said gastric secretion inhibitory subproteolytic enzymes are illustrated in the following Table stance and having a potency of at least five times that of III: an equal weight of whole dried gastric juice.

TABLE III Electro-phoretic Relative Milk Relative Pro- Heat Inactivation Migration Clotting Acteolytic Activity, at 0. Enzyme Optimal tivity, (PA)X Cg pH Paper Starch 2 CTXTT (PA) X0 pH20 pH30 cm. cm. CXT X z percent; percent Gastricsin 3. 0 o e. 5 43. 3 100 44. s 22.3 Human pepsim- 1. 52.0 9. 0 9. 34 44. 6 80.0 69. 0 11 2 Hog pepsin 2.0 9. 7 10.0 65. 6 66. 6 Rennin 100. O 8. 15

' 1 Using Spinco Durram type cell with pH 5.0 acetate butter of ionic strength 0.1 and the Spinco electrophoresis paper strips. The experiments were run with eight three cm. during 24 hours at 4 0.

strips at a constant field strength of volts,

2 Using Reco Model E-800-2 electrophoresis cell with pH 5.0 acetate bufier of ionic strength 0.1 the experimerits were run at a constant field strength of 4.55 volts/cm, during 22 ho urs at room tcmperat ure.

0 and T are the concentration and Clotting times measured according to the method of Berrldge.

4 (PAL, is the OD. at proteolytic activity 280 mu of the deproteinized solution from gastricsin assay, Cg is the concentration of gastricsin and C and (PA) the concentration and the 0.D. 280 mu from the deproteinized solutions from the assays of the respective enzymes.

of Anson and Mirsky.

Proteoly'tic activity is measured according to the method {The enzyme solutions were incubated for 10 minutes at the indicated temperature and the proteolytic activtty measured. The losses of activity were expressed as percent of Inactivation relative to that of the solution incubated at 45 0.

There is some indication that gastricsin is present in hog stomach and crude commercial preparations of pepsin.

While two examples of the preferred method embodying the present invention have been described along with the resultant products, it" will be apparent that many 2. The process as defined in claim 1 wherein the column is eluted successively with a series of aqueous inorganic buffered citrate solutions having pH values of about 3.0, 3.8, 4.2, and 4.6, and a series of aqueous in- 9 organic buttered phosphate solutions having pH values of about 5.3, 6.2, 7.0, and 8.5.

3. A process for isolating a water-soluble, protein-containing, gastric secretion inhibitory substance from gastric material of animal, including human, origin which comprises selectively adsorbing the water-soluble nondialyzable components of said gastric material on a column of carboxylic acid type cation-exchange resin equilibrated at about pH 3, eluting the column at an increasing pH value successively with a series of inorganic bufier solutions, said series including members having the approximate pH value of 6.15, 7.0 and 8.5, producing eluate having a pH gradually increasing from at least as low as that of the first member of said series to about 8.5, collecting separately eluate material obtained at pH from about 6.15 to about 8.5, dialyzing the said eluate material and lyophilizing the dialysate and recovering a concentrate of said gastric secretion inhibitory substance having a potency of at least five times that of an equal weight of whole dried gastric juice.

4. The process as defined in claim 3 wherein the column is eluted successively with a series of aqueous inorganic buffered solutions, chosen from the group consisting of: (l) citrate solutions having pH values of 3.0, 3.8, 4.2, and 4.6, and phosphate solutions having pH values of 5.3, 6.2, 7.0 and 8.5; and (2) phosphate solutions having pH values of 6.15, 7.0, and 8.5.

5. A process for the isolation of a pure proteolytic enzyme, gastricsin, from gastric material of animal, including human, origin which comprises adsorbing the Water-soluble non-dialyzable components of said gastric material on a column of carboxylic acid type cation-exchange resin equilibrated at a pH of about 3, eluting the column successively at an increasing pH value with a series of aqueous inorganic buffered solutions having pH values of about 3, 3.8, 4.2, and 4.6, said last three members of said series being applied successively as the efiiuent pH approaches that of the member next lowest thereto, producing eluate having a pH value gradually increasing from about 3 to greater than about 4.4, collecting separately the eluate fraction having a pH of about 4.4, and subjecting the said fraction to dialysis and lyophilization, whereby gastricsin is obtained.

6. The process as defined in claim 1 wherein the said separately collected fractions are dialyzed and then lyophilized.

7. The process as defined in claim 1 wherein the said gastric material is dialyzed and lyophilized human gastric juice.

8. The process as defined in claim 3 wherein said gastric material is dialyzed and lyophilized human gastric juice.

9. The product prepared by the process of claim 3.

10. The process as defined in claim 5 wherein the column is eluted with a series of aqueous inorganic bufiered citrate solutions.

11. The process as defined in claim 5 wherein the said dialyzed and lyophilized fraction is subjected to crystallization to obtain gastricsin in crystalline form.

12. The process as defined in claim 5 wherein the said dialyzed and lyophilized fraction is subjected to a crystallization procedure in the presence of ammonium sulphate whereby gastricsin is produced in crystalline form,

13. The process as defined in claim 5 wherein the said gastric material is human gastric juice which has been dialzed and lyophilized.

14. The product prepared by the process of claim 5.

15. The product prepared by the process of claim 6.

References Cited in the file of this patent UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,026,251 March 20, 1962 Stewart 6. Wolf, Jr. et a1. It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 1, line 48, for "components" read component column 2, line 32, for "factionating" read fractionating column 6, line 35, for "elute" read eluate col-mun 19, line 3O for "dialzed" read dialyzed line 32 for the claim reference numeral "6" read ll Signed and sealed this 10th day of July 1962.

(SEAL) Attest:

ERNEST w. swmza DAVID LADD Attesting Officer Commissioner of Patents 

3. A PROCESS FOR ISOLATING A WATER-SOLUBLE, PROTEIN-CONTAINING, GASTRIC SECRETION INHIBITORY SUBSTANCE FROM GASTRIC MATERIAL OF ANIMAL, INCLUDING HUMAN, ORIGIN WHICH COMPRISES SELECTIVELY ADSORBING THE WATER-SOLUBLE NONDIALYZABLE COMPONENTS OF SAID GASTRIC MATERIAL ON A COLUMN OF CARBOXYLIC ACID TYPE CATION-EXCHANGE RESIN EQUILIBRATAED AT ABOUT PH 3, ELUTING THE COLUMN AT AN INCREASING PH VALUE SUCCESSIVELY WITH A SERIES OF INORGANIC BUFFER SOLUTIONS, SAID SERIES INCLUDING MEMBERS HAVING THE APPROXIMATE PH VALUE OF 6.15, 7.0 AND 8.5, PRODUCING ELUATE HAVING A PH GRANDUALLY INCREASING FRO ATA LEAST AS LOW AS THAT OF THE FIRST MEMBER OF SAID SERIES TO ABOUT 8.5, COLLECTING SEPARATELY ELUATE MATAERIAL OBTAINED AT PH FROM ABOUT 6.15 TO ABOUT 8.5, DIALYZING THE SAID ELUATE MATERIAL AND LYOPHILIZING THE DIALYSATE AND RECOVERING A CONCENTRATE OF SAID GASTRIC SECRETION INHIBITORY SUBSTANCE HAVING A POTENCY OF AT LEAST FIVE TIMES THAT OF AN EQUAL WEIGHT OF WHOLE FRIED GASTIC JUICE.. 